1.1 Field of the Invention
The present invention relates to the field of stem cell culture media and to methods for culturing such cells. More particularly, the present invention provides methods and materials for culturing primate-derived primordial stem cells in a substantially undifferentiated state with and without a feeder layer. The present invention has applications in the areas of cell culture, tissue transplantation, drug discovery, and gene therapy.
1.2 The Related Art
Stem cells are cells capable of differentiation into other cell types having a particular, specialized function (xe2x80x9cfully differentiatedxe2x80x9d cells) or other types of stem cells which are capable of differentiation into a more narrow range of cell types (xe2x80x9cpluripotentxe2x80x9d cells). Stem cells having the ability to differentiate into any type of cell, i.e., pluripotent or fully differentiated, are called xe2x80x9ctotipotentxe2x80x9d. Such cells are also referred to as xe2x80x9cprimordial stem cellsxe2x80x9d. There has been great interest in isolating and growing primordial stem cells from primates, especially from humans, as such primordial stem cells could provide a supply of readily available cells and tissues of all types for use in transplantation, drug discovery, and gene therapy in humans.
Methods for isolating and growing primordial stem cells from primates have been described. Procedures for isolating and growing human primordial stem cells are described in co-pending U.S. patent application Ser. No. 08/829,372. Procedures for obtaining Rhesus monkey and other non-human primate primordial stem cells are described in co-pending U.S. patent applications Ser. Nos. 08/376,327; 08/591,246; 08/665,217; and WO 96/22362. Each of these patent applications is incorporated herein by reference in its entirety and for all purposes. In addition, methods for isolating Rhesus monkey primordial stem cells are described in Thomson et al. (1995 Proc. Natl. Acad. Sci. USA 92:7811-7848) also incorporated herein by reference in its entirety and for all purposes.
Unfortunately, current methods for growing primordial stem cells derived from primates in culture have not been as clearly defined as, and are relatively inefficient compared with, methods for culturing primordial stem cells for other species such as mouse. For example, current methods of culturing primate-derived primordial stem cells require a feeder layer that complicates and slows the process of cell cultivation. In addition, the formulation of an optimal culture media for propagating undifferentiated totipotent primate-derived primordial stem cells remains to be determined.
In particular, it is desirable to maintain cultures of totipotent primordial stem cells for extended periods or indefinitely. The ability to maintain cultures of undifferentiated, totipotent, primate-derived primordial stem cells for long periods facilitates the use of such cells for therapeutic purposes. Moreover, it would be desirable to grow cultures of substantially undifferentiated primate-derived primordial stem cells for periods sufficient to allow the production of primate-derived primordial stem cells having multiple genetic modifications for the production of tissues and for gene therapy.
The present invention provides methods and reagents for culturing primate-derived primordial stem cells in a substantially undifferentiated state. The methods and materials described herein provide improved culturing conditions that allow the preparation of primate-derived primordial stem cells having single or multiple genetic modifications. Such modified cells have important applications in gene therapy and tissue transplantation/implantation therapies.
In one aspect, the present invention provides a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state. In one embodiment, the cell culture medium of the invention comprises a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. This basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate selected from the group of feeder cells, such as mouse embryo fibroblast cells and STO cells, and an extracellular matrix derived from the feeder cells. The medium further includes non-essential amino acids, an anti-oxidant (for example, xcex2-mercaptoethanol), and, optionally, a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.
In more specific embodiments, the basic medium of the cell culture medium has an osmotic pressure of less than about 300 mOsm/kg. Still more specific embodiments are those for which the basic medium has an osmotic pressure of about 280 mOsm/kg. Yet other embodiments of the cell culture medium of the present invention include those for which the basic medium has an endotoxicity of less than about 0.1 endotoxin units per ml. More specific embodiments for which the endotoxicity of the basic medium is less than about 0.1 endotoxin units per ml are those embodiments for which the endotoxicity of the base medium is about 0.03 endotoxin units per ml.
In other embodiments the cell culture medium of the invention further includes a second growth factor. In a preferred embodiment, the second growth factor is selected from the group consisting of: Anti-IL-8, Anti-TGF-xcex25, Anti-BDNF, Anti-TNF-xcex2, Anti-VEGF, Anti-TGF-xcex2, IL-11, IL-6, IL-6+soluble IL-6 receptor, IL-1xcex1, IL-1xcex2, LIF, Anti-HB-EGF, IL-17, TFG-xcex2-1 LAP, MCP-1, bFGF, FGF-4, PDGF Soluble Receptor A, dexamethasone and Forskolin.
Suitable growth factors for use in the present invention can be determined using a method for screening for growth factors that is provided in another aspect of the present invention. According to one embodiment of this aspect of the invention, primate-derived primordial stem cells are grown using a cell culture medium of the present invention in the presence of a putative growth factor. A determination is made as to whether the putative growth factor enhances the growth of undifferentiated primate-derived primordial stem cells. Substances that enhance the growth of primate-derived primordial stem cells are classified as growth factors.
In another aspect, the present invention provides a culture of primate primordial cells, comprising at least one primate-derived primordial stem cell in fluid contact with the cell culture medium of the invention. Such cells can be human- or Rhesus-derived primordial stem cells, for example.
In still another aspect, the present invention provides methods for producing primate cell lines having one or more genetic modifications. According to one embodiment of this aspect of the present invention, primordial stem cells are grown using a cell culture medium of the invention. A first gene or nucleic acid is introduced into, or a first gene is modified in, these cells and a first clone population is derived. In a further embodiment, a second gene or nucleic acid is introduced into, or a second gene is modified in, the cells of the first clone population and a second clone population is derived. In some embodiments, the primordial stem cells are derived from human embryonic cells. In other embodiments the primordial stem cells are PSC43 cells, an aneuploid variant of Rhesus embryonic stem cells that is capable of growing in a feeder-free cell culture as described hereinbelow. This cell line has been found effective for screening for growth factors for cell culture media.
These and other aspects and advantages will become apparent when the Description below is read in conjunction with the accompanying Examples.
The present invention provides methods and materials for culturing primate-derived primordial stem cells in a substantially undifferentiated state and for identifying and quantifying undifferentiated primate-derived primordial stem cells. In addition, the present invention also provides screens for discovering substances that accelerate or retard the differentiation of such cells. In addition to the many benefits deriving from access to primate-derived primordial stem cells, the methods and materials provided by the present invention can be applied to produce primordial stem cells having single or multiple genetic modifications. Primate-derived primordial stem cells having such serial modifications have important applications, especially with respect to applications where euploid primate cells having genetic modifications are useful or required. Examples of such applications include, but are not limited to, the development of cell-based models, for primate, especially human, diseases, as well as the development of specialized tissues for transplantation to treat genetic diseases.
3.1 Definitions
The following terms will be defined as provided in this Section 3.1 unless otherwise stated. All other terminology used herein will be defined with respect to its usage in the particular art to which it pertains unless otherwise noted.
3.1.1 Basic Medium
Basic Medium refers to a solution of salts and nutrients that is effective to support the growth of primate-derived primordial stem cells in culture.
3.1.2 Conditioned Medium
Conditioned Medium refers to a growth medium that is further supplemented with soluble factors derived from feeder cells.
3.1.3 Embryonic Germ Cells
Embryonic Germ Cells or EG Cells are cells derived from the primordial germ cells of an embryo or fetus that are destined to give rise to sperm or eggs.
3.1.4 Embryonic Stem Cells
Embryonic Stem Cells or ES Cells are cells obtained from morula or blastocyst stages of a pre-implantation stage embryo.
3.1.5 Extracellular Matrix
Extracellular Matrix or Defined Matrix as used for the purposes of describing the present invention refers to one or more substances that provide substantially the same conditions for supporting cell growth as provided by the surfaces of feeder cells.
3.1.6 Feeder Cells
Feeder Cells as used for the purposes of describing the present invention refers to non-primordial stem cells on which primate-derived primordial stein cells are plated, which non-primordial stem cells provide a milieu conducive to the growth of the plated primate-derived primordial stem cells.
3.1.7 Growth Factor
Growth Factor as used for the purposes of describing the present invention refers to a substance that is effective to promote the growth of primordial stem cells that is not otherwise a component of the conditioned medium. Such substances include, but are not limited to, cytokines, chemokines, small molecules, neutralizing antibodies, and proteins.
3.1.8 Low Osmotic Pressure Medium
Low Osmotic Pressure Medium refers to a solution having an osmotic pressure of less than about 300 milli-osmols per kilogram (xe2x80x9cmOsm/kgxe2x80x9d).
3.1.9 Non-essential Amino Acids
Non-essential Amino Acids refers to the amino acids L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine.
3.1.10 Primordial Stem Cell
Primordial Stem Cell refers to either an embryonic stem cell or an embryonic germ cell as defined herein.
3.1.11 Primate-derived Primordial Stem Cell
Primate-derived Primordial Stem Cell refers to a primordial stem cell that is obtained from a primate species, including humans and monkeys, including genetically modified primordial stem cells obtained from a primate.
3.1.12 Pluripotent
Pluripotent refers to cells that are capable of differentiating into one of a plurality of different cell types although not necessarily all cell types. One example of pluripotent cells are bone marrow stem cells which are capable of differentiating into various blood cell types such as lymphocytes and red blood cells but not nerve cells. Thus, it will be recognized that while all totipotent cells are pluripotent, not all pluripotent cells are totipotent.
3.1.13 Substantially Undifferentiated
Substantially Undifferentiated refers to a group of primate-derived primordial stem cells of which at least about 50% are in an undifferentiated, totipotent, state.
3.1.14 Totipotent
Totipotent refers to cells that are capable of differentiating into any cell type including pluripotent and fully differentiated cells (i.e., cells no longer capable of differentiation into various cell types), such as, without limitation, bone marrow stem cells, cardiac muscle cells, astrocytes, or connective tissue cells.
3.2 Growing and Maintaining Primate-derived Primordial Stems Cells in a Substantially Undifferentiated State
As described in Sections 3.2.1 and 3.2.2 below, the present invention provides cell culture media, growth factors, and methods for growing and maintaining cultures of primate-derived primordial stem cells in a substantially undifferentiated state that provides for the growth and maintenance of totipotent primate-derived primordial stem cells for periods longer than heretofore available. The improved cell culture media of the invention can also be used to screen for additional growth factors and useful combinations of growth factors as described in Section 3.3 below. As discussed in Section 3.4 below, the ability to grow primate-derived primordial stem cells in a substantially undifferentiated, totipotent state using the improved cell culture media, growth factors, and methods provided herein provides important benefits including the ability to produce primate-derived primordial cell lines having single or multiple genetic modifications having important therapeutic applications.
3.2.1 Cell Culture Media for Growing and Maintaining Primate-derived Primordial Stem Cells in a Substantially Undifferentiated State
In one aspect, the present invention provides improved cell culture media for growing and maintaining primate-derived primordial stem cells in a substantially undifferentiated state. In one embodiment, the cell culture media of the present invention includes a low osmotic pressure, low endotoxin basic medium that is effective to support growth of primate-derived primordial stem cells; a nutrient serum effective to support growth of primate-derived primordial stem cells; a substrate selected from the group consisting of feeder cells, such as mouse (or other species) embryo fibroblast cells and STO cells, and an extracellular matrix derived from such feeder cells; non-essential amino acids; an anti-oxidant (reducing agent); and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.
In one particular embodiment, the osmotic pressure of the basic medium is less than about 300 milli-osmols per kilogram (xe2x80x9cmOsm/kgxe2x80x9d), and, more particularly, less than about 280 mOsm/kg. In one embodiment, the osmotic pressure of the basic medium is about 280 mOsm/kg. The endotoxicity, as measured in endotoxin units per milliliter (xe2x80x9ceu/mlxe2x80x9d) is less than about 0.1 eu, and, in a more particular embodiment, less than about 0.05 eu/ml. In a still more particular embodiment, the endotoxicity of the basic medium is less than about 0.03 eu/ml. In one particular embodiment, the endotoxicity of the basic medium is about 0.03 eu/ml. Methods for measuring endotoxicity are known in the art. For example, a preferred method is described in the xe2x80x9cGuideline on Validation of the Limulus Amebocyte Lysate Test as an End-product Endotoxin Test for Human and Animal Parental Drugs, Biological Products and Medical Devicesxe2x80x9d published by the U.S. Department of Health and Human Services, FDA, December 1987.
The nutrient serum can be any serum or serum-based solution that supplies nutrients effective to maintain the growth and viability of primate-derived primordial stem cells. Examples of such serum include, without limitation, fetal bovine serum (xe2x80x9cFBSxe2x80x9d) and fetal calf serum (xe2x80x9cFCSxe2x80x9d). In one embodiment, the serum is FBS. In a more particular embodiment, the FBS is provided in a concentration of between about 25% and about 1%. In a more particular embodiment, the FBS is provided in a concentration of between about 20% and about 2.5%. In as still more particular embodiment, the concentration of FBS in the cell culture medium is 20%. In another embodiment, the concentration of FBS is 2.5%.
Other embodiments of the cell culture media of the present invention include those for which a first growth factor includes one or more nucleosides. In more specific embodiments, the nucleoside(s) are selected from the group consisting of adenosine, cytosine, guanine, uridine and thymidine. Still more particular embodiments include those for which the nucleoside(s) selected are in about equal concentrations. More specific embodiments include those for which the concentration of nucleoside(s) included in the cell culture media of the invention is between about 0.1 xcexcM and about 30 xcexcM, and, more particularly, the media concentration is between about 0.3 xcexcM and about 10.0 xcexcM. In a still more particular embodiment, the concentration of nucleoside(s) is between about 0.5 xcexcM and about 5.0 xcexcM. In one embodiment, the concentration of nucleoside(s) is about 0.1 xcexcM. In still other embodiments, the first growth factor can be a pyruvate salt, such as sodium pyruvate or another pyruvate salt that is effective to maintain and/or enhance cell growth in a substantially undifferentiated state such as, for example, potassium pyruvate. The pyruvate salt can be combined with one or more of the above-described nucleosides. In one embodiment, the pyruvate salt is provided in a concentration of 1mM.
In some embodiments a second growth factor (as defined in Section 3.1.7) is also provided, again, to assist in the maintenance of cultures of primate-derived primordial stem cells in a substantially undifferentiated state. The identities and effective concentrations of such second growth factors can be determined using the methods described in Section 3.3 below or using techniques known to those of skill in the art of culturing cells. In one embodiment, a second growth factor is included with the cell culture media of the invention which second growth factor is selected from the group consisting of: Anti-IL-8, Anti-TGF-xcex25, Anti-BDNF, Anti-TNF-xcex2, Anti-VEGF, Anti-TGF-xcex2, IL-11, IL-6. IL-6+soluble IL-6 receptor, IL-1xcex1, IL-1xcex2, LIF, Anti-HB-EGF, IL-17, TFG-xcex2-1 LAP, MCP-1, bFGF, FGF-4, PDGF Soluble Receptor A, glucocorticoid (e.g., dexamethasone) and Forskolin. The second growth factor can be one or more of the above-listed substances as well as other growth factors that can be easily identified.
In one embodiment, the second growth factor is forskolin ([3R-(3xcex1, 4xcex1xcex2, 5xcex2, 6xcex2, 6axcex1, 10xcex1, 10xcex1xcex2, 10bxcex1)]-5-(acetyloxy)-3-ethenyldodecahydro-6, 10, 10b-trihydroxy-3, 4a, 7, 7, 10a-pentamethyl-1-H-naphtho[2, 1-b]pyran-1-one). In one embodiment, the forskolin is added to the cell culture medium of the invention to achieve a concentration of less than about 30 xcexcM. In a more particular embodiment, the concentration of forskolin in the cell culture medium of the invention is between about 5 xcexcM and about 15 xcexcM, and, more particularly, between about 8 xcexcM and about 12 xcexcM. In one embodiment, the concentration of forskolin added to the cell culture medium of the invention is about 10 xcexcM. In another embodiment, the concentration of forskolin is about 20 xcexcM.
In another embodiment, the second growth factor is selected from the group consisting of xe2x80x9cbasicxe2x80x9d FGF (xe2x80x9cbFGFxe2x80x9d) and/or FGF-4, alone or in combination with human insulin, anti-TGF-xcex2-1 antibody, and EGF. In one embodiment, the concentration of bFGF in the cell culture medium is about 5 nanograms/milliliter (xe2x80x9cng/mlxe2x80x9d), either alone or combined with human insulin. When combined with bFGF, the concentration of human insulin is about 8 xcexcg/ml. In those embodiments for which EGF is added to the cell culture medium of the invention, the concentration of EGF is about 0.1 ng/ml.
The cell culture media of the invention also includes an anti-oxidant (reducing agent), such as xcex2-mercaptoethanol. In a preferred embodiment, the xcex2-mercaptoethanol has a concentration of about 0.1 mM. Other anti-oxidants such as monothioglycerol or dithiothreitol (xe2x80x9cDTTxe2x80x9d), alone or in combination, can be used to similar effect. Still other equivalent substances will be familiar to those of skill in the cell culturing arts.
In addition to the above-described components, the cell culture media of the invention further includes a substrate selected from the group consisting of feeder cells, such as mouse (or other species) embryo fibroblast cells and STO cells, and an extracellular matrix derived from such feeder. In one embodiment, mouse embryo fibroblasts obtained from dissection of 13.5-day-old CF-1 strain mice are used. Other suitable feeder cell lines will be familiar to those of skill in the cell culture art. If feeder cells are used, as opposed to extracellular matrix, the cells can be mitotically inactivated (e.g., by irradiation or chemically) to prevent further growth and seeded on plates. The primate-derived primordial stem cells can then be grown on the plate in addition to the feeder cells. Alternatively, the feeder cells can be first grown to confluence and then inactivated to prevent further growth. It will be appreciated that such an approach has the advantage of simplifying the management of the cell culture as the growth of only one set of cells, the primordial stem cells, need only be monitored.
Not wishing to be bound to any theory, it is believed that the use of such feeder cells, or an extracellular matrix derived from such feeder cells, provides one or more substances necessary to promote the growth of primate-derived primordial stem cells and/or prevent or decrease the rate of differentiation of such cells. Such substances are believed to include membrane-bound and/or soluble cell products that are secreted into the surrounding medium by the cells. Thus, those of skill in the cell culturing arts will recognize that additional cell lines can be used with the cell culture medium of the present invention to equivalent effect and that such additional cell lines can be identified using standard methods and materials. In addition, those of skill will also recognize that one or more substances produced by the feeder cells, or contained in the extracellular matrix, can be identified and added to the cell culture medium of the invention to obviate the need for such feeder cells and/or such extracellular matrix.
In one particular embodiment of the invention, the preparation of which is described in detail in Section 4.1 below, a cell culture medium provided by the present invention includes the components and concentrations set forth in Table 1.
3.2.2 Growing Primate-derived Primordial Stem Cells Using the Cell Culture Media of the Invention
In another aspect, the present invention provides methods for growing primate-derived primordial stem cells in a substantially undifferentiated state and cultures of such cells in such cell culture media described above in Section 3.2.2. Detailed examples of the methods provided by the present invention can be found in Sections 4.1 and 4.2 below.
The primate-derived primordial stem cells to be cultured can be obtained using known methods and materials. Procedures for isolating human primordial stem cells are described in co-pending U.S. patent application Ser. No. 08/829,372, filed on Mar. 31, 1997. Procedures for obtaining Rhesus monkey and other non-human primate primordial stem cells are described in co-pending U.S. patent application Ser. Nos. 08/376,327, filed Jan. 20, 1995; 08/591,246, filed Jan. 18, 1996; WO 96/22362, published Jul. 25, 1996; 08/665,217, filed Jun. 14, 1996; and 08/874,695, filed Jun. 13, 1997. Each of these patent applications is incorporated herein by reference in their entirety and for all purposes. In addition, methods for isolating Rhesus monkey primordial stem cells can be found in Thomson et al. (1995 Proc. Natl. Acad. Sci. USA 92:7844-7848) also incorporated herein by reference in its entirety and for all purposes.
Once isolated, the primate-derived primordial stem cells are cultured using the above-described conditioned medium using any of a variety of techniques. In one embodiment, a container holds feeder cells in a non-conditioned medium. A matrix of lysed feeder cells is prepared using standard methods. One example of the preparation of such a matrix is provided in Section 4.2 below. The primordial stem cells to be cultured are then added atop the matrix along with the conditioned medium. Alternatively, the primate-derived primordial stem cells can be grown on living feeder cells using methods known in the cell culture arts. The growth of the primordial stem cells is then monitored to determine the degree to which the cultured cells have become differentiated. In one embodiment, described in Section 4.2 below, a marker for alkaline phosphatase is used to ascertain which cells have differentiated. When a sufficient number of cells have differentiated, or when the culture has grown to confluence, at least a portion of the undifferentiated cells is passaged. The determination to passage the cells and the techniques for accomplishing such passaging can be performed using standard techniques.
3.3 Screens for Growth Factors
In another aspect, the present invention provides screens for determining growth factors that promote or inhibit the differentiation of primate-derived primordial stem cells in culture. In one embodiment, an aneuploid variant of Rhesus 278.5 ES cells having 43 chromosomes, hereinafter referred to as xe2x80x9cPSC43 cellsxe2x80x9d, is used as a primary screen to identify substances that promote the growth of primate-derived primordial stem cells in a substantially undifferentiated state. In one embodiment of the primary screen, the presence of increased alkaline phosphatase activity indicates that the substance being tested is a growth factor. Substances that are found to produce statistically significant promotion of the growth of PSC43 cells in an undifferentiated state can then be tested against normal primate-derived primordial embryonic stem cells. Substances found to be effective growth factors for these cells are then tested in combinations to determine the presence of any synergistic effects. Optionally, a secondary screen can be employed to confirm growth factors identified by the primary screen.
In one embodiment, described in detail in Section 4.4.2, the screens are performed on groups of PSC43 cells grown under four different growth conditions (alternatively, normal Rhesus, human or other primate-derived primordial stem cells can be used). A first growth condition (described in Section 4.4.2.1) includes STO (or other suitable) feeder cells in the medium described in Section 3.2.1 above. A second growth condition (Section 4.4.2.2) includes growing cells under the same conditions as the first growth condition, except that an extracellular matrix of STO or MEF cells is used in place of the feeder cells (see Section 3.2.1). A third growth condition (Section 4.4.2.3) includes growing cells on a xe2x80x9cdefined matrixxe2x80x9d that comprises one or more substances that approximate the extracellular matrix of feeder cells. In one embodiment, the defined matrix includes one or more substances selected from the group consisting of collagen II, heparan sulfate, and merosin. Still other suitable substances can be determined using methods known in the cell culturing arts. A fourth growth condition (Section 4.4.2.4) includes growing cells under the same conditions as the first growth condition with the exception that 2.5% FBS is used in the nutrient serum instead of 20% FBS.
In one embodiment, the level of expression of alkaline phosphatase is determined for each group of cells exposed to a particular putative growth factor using the methods described herein (see Section 4.2). Substances that are correlated with increased alkaline phosphatase expression relative to unexposed control cells are considered to be growth factors. In a particular embodiment, substances found to produce an increase of alkaline phosphatase expression greater than about 20% as compared with the control are considered growth factors.
In another embodiment, substances identified as growth factors in the primary screen are tested in a secondary screen to determine the presence or absence of a correlation between exposure of the cells to the substance and a parallel increase in the expression of surface markers associated with lack of differentiation such as telomerase (described below in section 4.7), stage-specific embryonic antigen-4 (SSEA-4), stage-specific embryonic antigen-3 (SSEA-3) (both described by Kannagi et al., EMBO J, 1983, 2(12):2355-61), TRA-1-60 antigen and TRA-1-81 antigen (both described by Andrews et al., Hybridoma, 1984, 3(4) 347-61.
In such an embodiment, the cells are cultured as described in the primary screen. The cells are then exposed to an antibody raised against one or more of the surface marker(s) being screened, and/or the presence or absence of telomerase expression in the exposed cells is determined (see Section 4.7). In some embodiments, the surface marker antibodies are incubated with a second antibody coupled with a reporter such as a fluorescent label so that cells expressing the appropriate antigenic marker are rendered fluorescent. Labeled cells can then be sorted and counted using standard methods, e.g., a fluorescence-activated cell sorter (xe2x80x9cFACSxe2x80x9d). The numbers of labeled and unlabelled cells can then be compared to determine the effect of the putative growth factor. Alternatively, following exposure to unlabelled cell surface marker antibodies, the cells can be exposed to a second antibody that is specific for the cell surface marker antibody in an ELISA (Enzyme-Linked ImmunoSorbent Assay) format from which the number of cells expressing the desired surface antigen can be quantitated calorimetrically or by measurement of fluorescence. Still other methods of quantitating cells expressing surface antigens will be familiar to those having skill in the cell culture arts.
Substances identified as growth factors in the primary, and, optionally, secondary, screens are screened again using the same format as the primary screen discussed above but wherein actual primate-derived primordial stem cells are used. Those substances that are confirmed to be growth factors are then tested in combination (e.g., combinations of two or three substances) to determine the presence of any synergistic properties among the growth factors. In addition, substances that may promote differentiation or retard the growth of undifferentiated cells can be identified. For example, antibodies directed to substances in the growth medium can be added to prevent those substances from interacting with the cells being cultured.
One example of the use of the above-described techniques for determining an optimized culture medium is provided in Section 4.5 below in which alkaline phosphatase (AP) activity is used as marker for undifferentiated cells. There, PSC43 cells were grown using an STO-based extracellular matrix in a medium that included DMEM with 4.5 g/L glucose, 0.1 mM non-essential amino acids, 0.1 mM xcex2-mercaptoethanol and 20% fetal bovine serum. To this medium sodium pyruvate, adenosine, guanosine, thymidine, cytidine, uridine, phenol red dye, and HEPES buffer were added to determine the effect of each substance individually. As described in detail in Section 4.2.1, it was determined that the addition of 1 mM sodium pyruvate to the medium resulted in an increase in the amount of AP activity. In addition a final concentration of 1 xcexcM each of adenosine, guanosine, thymidine, cytidine and uridine to the sodium pyruvate growth medium resulted in even more enhanced AP activity levels of PSC 43 cells at both the 11th and 17th passages. The use of anti-retinoic acid antibodies to deplete the growth medium of retinoic acid also provided enhanced growth of undifferentiated cells as measured by AP activity as described in Section 4.6.
Substances identified as promoters of undifferentiated cell growth using the above-described screening methods and materials are described in Table 2 below. Those having skill in the cell culture arts will recognize that several factors identified as promoters of undifferentiated cell growth are members of the IL-6 and LIF families of cytokines. Such substances have been recognized as interacting with specific receptors that heterodimerize with gp 130 to effect signal transduction (Fourcin, et al., J. Biol. Chem., 271(20):11756-11760 (1996)) and, thereby, maintenance of undifferentiated growth. Unfortunately, IL-6 and LIF-family receptors are highly species specific; therefore, growth factors isolated from one species may not function with cell lines isolated from another species. However, anti-gp130 antibodies can also be used to effect signal transduction by gp130 (Wijdenes, et al., Eur. J. Immunol., 25:3474-3481). Thus, the present invention further includes methods and media for culturing primate-derived primordial stem cells including anti-gp130 antibodies.
In another embodiment, the cell culture methods and materials of the invention include a glucocorticoid, such as dexamethasone ((11xcex2, 16xcex1)-9-fluoro-11, 17, 21-trihydroxy-16-methylpregna-1, 4,-diene-3, 20-dione). In one embodiment, the dexamethasone is provided at a concentration of between about 1.0 nanomolar (nM) and about 10.0 xcexcM. In one more particular embodiment, the concentration of dexamethasone is between about 1.0 nM and about 1.0 xcexcM. In another particular embodiment, the concentration of dexamethasone is between about 1.0 nM and about 500 nM. In still another embodiment, the dexamethasone is provided at a concentration of between about 1.0 nM and about 100 nM. In yet another embodiment, the dexamethasone is provided at a concentration of about 10 nM. In still other embodiments, the dexamethasone is combined with at least one substance that is a member of the IL-1, IL-6, IL-11, or LIF families of cytokines. In some embodiments, dexamethasone is combined with at least one of the following: IL-1xcex2 (at a concentration of about 50 picograms/mi (pg/ml)), IL-6 (at a concentration of about 0.004 micrograms/ml (xcexcg/ml)), LIF (at a concentration of about 1.2 ng/mil), and IL-11 (at a concentration of about 1.0 ng/ml).
3.4 Applications of the Cell Culture Growth Media of the Invention
The improved cell culture media and methods for growing primate-derived primordial stem cells in a substantially undifferentiated state that is provided by the present invention will be seen to be applicable to all technologies for which primate-derived cell lines are useful. Of particular importance is the use of the cell culture media and methods of culturing primate-derived primordial stem cells provided by the present invention to create new primate primordial stem cell lines having single or multiple genetic modifications which application is discussed in Section 3.4.1. Cells produced using the media and methods of the present invention can be mounted on surfaces to form biosensors for drug screening (see Section 3.4.2). In addition the observation that primate primordial stem cells are telomerase positive can be used to determine the engraftment potential of primordial stem cells, both primate-derived and non-primate-derived, as described in Section 3.4.3.
3.4.1 Creation of Primate-derived Primordial Stem Cells Cell Lines Having Multiple Genetic Modifications
In one aspect, the methods and culture media of the present invention are used to produce primate-derived primordial stem cells having single or multiple genetic modifications. Genetic alteration of cells is desirable for many reasons, such as providing modified cells for gene therapy and replacement tissues for grafting or implantation (e.g., to avoid host rejection of the cells).
According to one embodiment of this aspect of the present invention, primordial stem cells are grown using the culture media and methods described in Section 3.2.1 above. A first gene is modified in, or introduced into, at least one of the cells of the cell culture and from the resulting culture a first clone population of modified primordial stem cells is derived. The first clone population can be grown in the culture media of the invention allowing the establishment of a cell line with the desired genetic modification. If further genetic modifications are needed, a second gene is modified in, or introduced into, at least one cell of the first clone population to produce a second clone population having first and second genetic modifications. Alternatively, the first and second genetic modifications can be introduced into the same primordial stem cell with subsequent simultaneous screening for both modifications (i.e., circumventing the need to isolate a first clone population); however, the preferred procedure is a stepwise procedure.
The methods used to perform the genetic modifications to the cells can be any of those known in the molecular biological arts for making genetic transforms. Such methods include, but are not limited to, the use of positive-negative selector vectors as described in U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059; and 5,631,153 to Capecchi, et al.; and U.S. patent application Ser. No. 08/781,559. In addition, yeast artificial chromosomes (YACs) can be employed to perform genetic modifications as described in U.S. patent application Ser. Nos. 08/597,532; 08/397,547; 08/187,161; 08/276,565; 08/375,482; 08/485,505; and 08/372,482. Furthermore, isogenic DNA constructs can be used with the primordial stem cells cultured using the methods and materials provided by the present invention as described in U.S. patent application Ser. No. 08/563,138. Still other methods include those described in U.S. Pat. No. 5,591,625 to Gerson, et al. for the preparation stem cells capable of augmented expression of certain gene products, signal transduction molecules, cell surface proteins and the like for therapeutic applications. U.S. Pat. No. 5,583,016 describes methods for introducing a recombinant gene for the RNA component of telomerase into cells and GB2317891 describes methods for increasing the amount of human telomerase reverse transcriptase (hTRT) in a cell, for example, by introducing the reverse transcriptase subunit of telomerase into cells. These patents and patent applications are incorporated herein by reference in their entirety and for all purposes.
As is apparent to one of ordinary skill in the art, altered expression of gene products can be achieved by modifying the coding sequence of a gene product or altering flanking regions of the coding sequence. Thus, as used herein, the term xe2x80x9cgenetic modificationxe2x80x9d includes alterations to the sequence encoding a gene product, as well as alterations to flanking regions, in particular the 5xe2x80x2 upstream region of the coding sequence (including the promoter). Similarly, the term xe2x80x9cgenexe2x80x9d encompasses the coding sequence and regulatory sequences that may be present flanking the coding sequence, as well as other sequences flanking the coding sequence. In addition, as is known in the art, genetic modifications can be achieved by introducing a nucleic acid that does not necessarily comprise the entire gene sequence into the cell, e.g., by introducing a nucleic acid that can be inserted into the genome by recombination.
In one embodiment of the invention in which genetically-modified primate-derived primordial stem cells are to be use for implantation into a patient, e.g., to treat Parkinson""s disease, the primate-derived primordial stem cells are modified genetically to express Fas ligand (also known as CD95). Cells expressing the Fas ligand are known to induce apoptosis in T cells; thereby becoming immunologically privileged (Griffith, et al., Science, 270:1189-1192 (1995); Bellgrau, et al., Nature, 377:630-632 (1995)). In one embodiment, the present invention provides primate-derived primordial stem cells having multiple genetic modifications wherein at least one of the modifications is the expression of Fas ligand. In another embodiment, the modified primate-derived primordial stems cells are differentiated into a different cell type using, e.g., a differentiation promoter listed in Table 3 below.
3.4.2 Biosensors Comprising Primate-derived Primordial Stem Cells
In another aspect, cells cultured and/or modified using the materials and methods provided by the present invention are mounted to support surfaces to screen for bioactive substances. In one embodiment, the cells are coupled with a substrate such that electrophysiological changes in the cells in response to external stimuli can be measured, e.g., for use as a high-throughput screen for bioactive substances. In one more particular embodiment, the cells have been transfected with DNA that targets, expresses, or knocks-out specific genes or gene products in the cell. By providing such chip-mounted cells coupled with measuring devices, such as a computer, many compounds can be screened rapidly and accurately. The biosensors could also be coupled to the measuring device in arrays for large-scale parallel screening.
In another embodiment, a reporter gene is incorporated into the DNA of a primordial stem cell that is functionally coupled with a copy of a gene associated with a particular disease state (e.g., BRCA-1 in the case of breast cancer) using the methods described in Section 3.4.1 above. In one embodiment, the reporter is sensitive to both transcription and post-transcriptional events. The primordial stem cells are allowed to differentiate such that the differentiated progeny each contain one copy of the disease gene/reporter construct. The cells are then screened against putative therapeutic agents. This allows the correlation of gene expression and responsiveness to a potential therapeutic agent with the state of differentiation of the cell. By suitable selection of the reporter, such a screening strategy can be executed with the above-described high-throughput biosensors. Still other applications of biosensors such as discussed herein will be apparent to those having skill in the art.
3.4.3 Prediction of Stem Cell Engraftment Potential of Primate-derived Primordial Stem Cells
In yet another aspect, the determination of telomerase activity as a marker for cell differentiation as described in Section 4.7 below is used to determine the engraftment potential of primate-derived primordial stem cells cultured using the methods and materials of the present invention. In one embodiment, primordial stem cells cultured using the methods and materials of the invention are allowed to differentiate, or, alternatively, induced to differentiate, to produce pluripotent daughter cells such as hematopoietic stem cells for use in transplantation. Induction of differentiation can be performed using agents effective to induce differentiation such as retinoic acid. The cells may be genetically unaltered or may be genetically modified using the methods described in Section 3.4.1 above. The pluripotent daughter cells identified as having strong telomerase expression can be specifically isolated and used for transplantation or further culturing and/or modification as described above.
In another embodiment, the use of the cell culture medium and methods of the present invention to provide cultures of unmodified and modified primate primordial stem cells is used to screen for substances that improve the monitoring of stem cells or the collection of stem cells. For example, putative engraftment enhancing substances can be added to a cell culture grown using the methods described above. Substances that increase telomerase expression compared to a control cell culture that lacks the putative enhancing substance are identified as engraftment promoters or enhancers.